ABSTRACT Alcohol use disorder (AUD) is characterized by inflexible compulsive drinking despite negative consequences. This behavioral inflexibility is associated with deficits in reversal learning. The thalamus and the dorsomedial striatum (DMS) are critical reversal learning in flexible behavior. The DMS contains principal medium spiny neurons (MSNs). MSNs are located either in the direct-pathway (dMSNs) or indirect-pathway (iMSNs) of the basal ganglia. Both dMSNs and iMSNs receive cortical inputs and positively and negatively regulate the selection of ?Go? actions, respectively. The DMS also contains cholinergic interneurons (CINs). CIN release acetylcholine to modulate MSN activity. During reversal learning, thalamic inputs excite DMS CINs to generate burst-pause firing and stop ongoing actions. It is not known how thalamically evoked CIN activity regulates MSNs in AUD and contributes to inflexible behavior. The goal of this application is to study how excessive ethanol intake alters thalamostriatal transmission, leading to behavioral inflexibility. Our long-term objective is to develop new therapeutics to restore behavioral flexibility and treat AUD. The hypothesis, based in part on applicant?s preliminary results, is that excessive ethanol intake compromises thalamic regulation of DMS CINs and CIN-mediated regulation of cortical inputs onto dMSNs and iMSN, leading to inflexible behaviors, which can be counteracted by optogenetic excitation of thalamostriatal transmission onto CINs. We will test this hypothesis by pursuing the following three specific aims. (1) Investigate whether excessive ethanol intake reduces the burst-pause response of DMS CINs and acetylcholine release from CINs. (2) Determine whether excessive ethanol intake compromises CIN-mediated regulation of corticostriatal transmission in DMS MSNs. And (3) evaluate the ability of optogenetic excitation of thalamic inputs onto DMS CINs to improve reversal learning in animals with a history of excessive ethanol intake. This research is conceptually innovative because it focuses on the relatively neglected area of the cholinergic contribution to AUD. It is technically innovative in its use of combined genetically encoded acetylcholine sensors, dual-channel optogenetics, and rabies- mediated expression systems at a synapse with defined pre- and postsynaptic sites to determine the cholinergic contribution to ethanol-mediated inflexibility. These essential research questions cannot be addressed using conventional methodologies. Knowledge generated from this proposal will provide novel strategies for reversing inflexible behavior and thereby reduce excessive ethanol intake in AUD.